A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Bovara, Roberto; Carrea, Giacomo; Riva, Sergio; Secundo, Francesco

doi:10.1007/bf00142949

Cited by 24 publications

(17 citation statements)

References 15 publications

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“…An electrophoretically homogeneous sample with a main protein band of about 27 kDa in SDS-PAGE analysis was prepared by subsequent hydrophobic interaction chromatography and was subjected to kinetic determinations ( Table 1, entries [8][9][10][11][12]. This HSDH showed a higher specificity for the substrates of the oxidative reactions, cholic acid (1) and 12-ketochenodeoxycholic acid (4), being practically equivalent both in terms of K m and k cat /K m values.…”

Section: Resultsmentioning

confidence: 99%

“…NADP(H) in situ regeneration in the two steps was carried out with aketoglutarate/glutamate dehydrogenase and glucose/ glucose dehydrogenase, respectively. [8] All these enzymatic activities were NADPH-dependent, this hampering their concurrent use in a single-step biotransformation of cholic acid (1) to 3. In fact, a mixture of 1, 3 and of the intermediate products 3a-hydroxy-7,12-dioxo-5b-cholanoic acid (2), 12-ketochenodeoxycholic acid (3a,7a-dihydroxy-12-oxo-5b-cholanoic acid, 4), 3a,12a-dihydroxy-7-oxo-5b-cholanoic acid (5), and ursocholic acid (3a,7b,12a-trihydroxy-5b-cholanoic acid, 6, see Scheme 2) is expected to occur in the absence of a suitable driving force leading the synthesis towards the desired product.…”

Section: Introductionmentioning

confidence: 99%

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One‐Pot Multienzymatic Synthesis of 12‐Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row

et al. 2009

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Abstract:The hydroxysteroid dehydrogenases (HSDHs)-catalyzed one-pot enzymatic synthesis of 12-ketoursodeoxycholic acid (3a,7b-dihydroxy-12-oxo-5b-cholanoic acid), a key intermediate for the synthesis of ursodeoxycholic acid, from cholic acid has been investigated. This goal has been achieved by alternating oxidative and reductive steps in a onepot system employing HSDHs with different cofactor specificity, namely NADH-dependent HSDHs in the oxidative step and an NADPH-dependent 7b-HSDH in the reductive one. Coupled in situ regeneration systems have been exploited not only to allow the use of catalytic amounts of the cofactors, but also to provide the necessary driving force to opposite reactions (i.e., oxidation and reduction) acting on different sites of the substrate molecule. Biocatalysts suitable for the set-up of this process have been selected and their kinetic behaviour in respect of the reactions of interest has been evaluated. Finally, the process has been studied employing the enzymes both in free and compartmentalized form.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

One‐Pot Multienzymatic Synthesis of 12‐Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row

et al. 2009

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“…When prepared chemically the yield was very low (less than 30%) [218]. When a mixed enzymatic-chemical procedure was used it required chromic anhydride which is a strong environment pollutant [219]. A two-step enzymatic procedure, Fig.…”

Section: Redox Reactions Involving Steroidsmentioning

confidence: 99%

Redox Enzymes Used in Chiral Syntheses Coupled to Coenzyme Regeneration

Leonida¹

2001

CMC

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Due to their ability to perform reactions in a stereo- and regiospecific manner, while functioning under mild conditions of temperature and pH, enzymes are increasingly used for chiral synthesis in the pharmaceutical industry, in medicinal chemistry, agriculture, cosmetic and food industry. The oxidoreductases as catalysts require a coenzyme (cofactor) which functions as an electron carrier. If used in stoichiometric amounts the cost of coenzymes makes synthetic applications of redox enzymes prohibitively expensive for industrial applications. The present review updates previous discussions about the objectives of cofactor regeneration as a requirement for the applicability of enzymatic redox reactions, and about the strategies customarily used to this end. Different types of chiral syntheses coupled successfully to coenzyme regeneration (amino acid synthesis, lactone synthesis, synthetic reactions involving alcohols, hydroxy acid and steroid synthesis, deracemization) are then discussed. New catalysts, cross-linked enzyme crystals, prepared from redox enzyme are presented in a small paragraph together with some of their applications. Special attention is given to whole cells used in this type of synthesis and their advantages and disadvantages are presented in one paragraph. Finally, different reactors, within which were conducted chiral syntheses catalyzed by oxireductases and coupled to cofactor regeneration, are briefly discussed.

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“…An important target compound in the synthesis of ursodeoxycholic acid is 12-ketoursodeoxycholic acid, which can be converted by a Wolff-Kishner reduction into ursodeoxycholic acid. One route for the synthesis of 12-ketoursodeoxycholic acid starts from cholic acid (3α,7α,12α-trihydroxy-5β -cholanoic acid), which can be converted by two oxidative steps catalyzed by 7α-and 12α-hydroxysteroid dehydrogenases (HSDHs), followed by a 7β -HSDHcatalyzed reductive step [5,6]. A further route starts from 7-ketolithocholic acid which is converted into ursodeoxycholic acid by reducing the 7-keto group by 7β -HSDH [7,8].…”

Section: Introductionmentioning

confidence: 99%

Large-scale Enzymatic Synthesis of 12-Ketoursodeoxycholic Acid from Dehydrocholic Acid by Simultaneous Combination of 3α-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni and 7β-Hydroxysteroid Dehydrogenase from Collinsella aerofaciens

Bakonyi

Wirtz

Hummel

2012

Zeitschrift Für Naturforschung B

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Dedicated to Professor Heribert Offermanns on the occasion of his 75 th birthday12-Keto-UDCA is an important optically active component for the drug ursodeoxycholic acid (UDCA). Starting from the three-keto compound dehydrocholic acid, the carbonyl groups at position 3 and 7 have to be reduced stereo-and regioselectively. In this case we applied two hydroxysteroid dehydrogenases for this purpose, the NAD-dependent 3α-HSDH from Pseudomonas testosteroni and the NADP-dependent 7β -hydroxysteroid dehydrogenase from Collinsella aerofaciens. Both enzymes can be produced in high yields by an Escherichia coli strain as recombinant proteins. In order to avoid impurities by the 7α-hydroxysteroid dehydrogenase of Escherichia coli, a mutant strain with an inactivated 7α-enzyme was applied for producing the three enzymes. For bioconversion, the dehydrogenases can be used as crude enzyme samples and are applied simultaneously. A 1.8 L batch of 100 mM DHCA incubated at pH = 8.0 and 25 • C resulted in 80 g crude product with a quite high purity of ≥ 99.5 % as judged by HPLC analysis.

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A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Cited by 24 publications

References 15 publications

One‐Pot Multienzymatic Synthesis of 12‐Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row

One‐Pot Multienzymatic Synthesis of 12‐Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row

Redox Enzymes Used in Chiral Syntheses Coupled to Coenzyme Regeneration

Large-scale Enzymatic Synthesis of 12-Ketoursodeoxycholic Acid from Dehydrocholic Acid by Simultaneous Combination of 3α-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni and 7β-Hydroxysteroid Dehydrogenase from Collinsella aerofaciens

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